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Potravinarstvo Slovak Journal of Food Sciences SURVEY OF Potravinarstvo Slovak Journal of Food Sciences Potravinarstvo Slovak Journal of Food Sciences vol. 14, 2020, p. 721-728 https://doi.org/10.5219/1322 Received: 10 February 2020. Accepted: 22 April 2020. Available online: 28 September 2020 at www.potravinarstvo.com © 2020 Potravinarstvo Slovak Journal of Food Sciences, License: CC BY 3.0 ISSN 1337-0960 (online) SURVEY OF MYCOBIOTA ON SLOVAKIAN WINE GRAPES FROM SMALL CARPATHIANS WINE REGION Soňa Felšöciová, Miroslava Kačániová, František Vrábel ABSTRACT A total of 13 samples of grapes (bunches) without apparent fungal contamination were analyzed. The samples were collected during the 2019 harvest from Vrbové village in the Small Carpathian region of Slovakia. For the isolation of fungi were used the direct plating technique on DRBC plates. The plates were incubated aerobically at 25 ±1 °C for one week in the dark. The data obtained from the cultivation of the grape berry samples revealed a high diversity of fungal species (a total of 1044 isolates were obtained). Alternaria and Rhizopus were the main components of the wine grape mycobiota of the Vrbovský subregion at harvest time (92%, each), followed by Cladosporium (85%), Penicillium (77%), Botrytis and Epicoccum (54%, each). The most abundant genera found by descending order were Penicillium (25%), Alternaria (24%), Cladosporium (20%), and Rhizopus (12%) and only in minor percentage by Aspergillus (3%) among others. The main fungal species isolated from genera Penicillium and Aspergillus were Penicillium expansum (57% RD) and A. section Nigri (97% RD). Of 17 analyzed Penicillium strains, 65% were able to produce at least one of the six mycotoxins analyzed in in vitro conditions by means of thin-layer chromatography method: citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, and roquefortin C. Keywords: grape; filamentous fungi; Penicillium; mycotoxin; Slovakian vineyard INTRODUCTION development in wine grapes brings significant yield losses Viticulture is an important activity in many countries for winemaking, alters the chemical composition of wine (Einloft et al., 2017). Vine growing and viticulture have grapes, and produces secondary fungal metabolites and a very long tradition in Slovakia and are parts of the enzymes that together adversely affect wine flavor and country's cultural and historical heritage. Hundreds of color as well as yeast and lactic acid bacteria growth years of viticulture and viniculture have created a specific during vinification (Fleet, 2003). Among them, it is of type of landscape (Bezák et al., 2010), with unique great concern the presence of toxicogenic fungi in wine cultural and aesthetic values (Salašová and Štefunová, grapes capable of producing mycotoxins that could persist 2009). In total there exist six viticultural regions in during the winemaking process up to wine, being a high Slovakia with forty areas and wine-growing villages risk for consumer's health (Paterson et al., 2018; Prendes (ÚKSÚP, 2019a). Slovakia features almost 660 producers et al., 2015). growing around 13.500 ha of vines (of a potential The genus Alternaria is ubiquitously distributed and 15.300 ha) (ÚKSÚP, 2019b) for a production of about includes both saprophytic and opportunistic plant- 300.000 hL annually, which is primarily sold within the pathogenic species, which may affect crops in the field or national market. cause harvest and postharvest decay of plant products. The microflora of the grapes is highly variable, mostly Moreover, several Alternaria species are known to due to the influence of external factors as environmental produce toxic secondary metabolites, Alternaria parameters, geographical location, grape cultivars, and mycotoxins. The major Alternaria mycotoxins are the application of phytochemicals on the vineyards tetramic acid derivate, tenuazonic acid, and the (Pretorius, 2000; Pinto et al., 2014). A variety of fungal dibenzopyrone derivates, alternariol (AOH), and genera, mainly Botrytis, Alternaria, Aspergillus, alternariol monomethyl ether (AME) (Prendes et al., Penicillium, and Cladosporium, can contribute to grape 2015). Despite the toxic effects of the Alternaria toxins spoilage before harvest (Bellí et al., 2006; Magnoli et al., and their documented occurrence, they have not yet 2003; Medina et al., 2005). Filamentous fungi impact received the same attention as others mycotoxins and up to negatively in the production, sensory quality, and safety now, there is no regulation about them (EFSA, 2011). As characteristics of the wine in several ways. Their an opportunistic pathogen, it has the potential to cause a grape berry rot in the field under high disease pressure Volume 14 721 2020 Potravinarstvo Slovak Journal of Food Sciences situations. Strikingly, Alternaria has not been extensively colonies were identified to species level according to studied in wine grapes as a hazardous genus. micro and macroscopic criteria, using the keys of Klich Penicillium has gained attention as grapevine pathogens. (2002) and Pitt and Hocking (2009). Penicillium strains Penicillium expansum can cause rot in grapes, but does not were isolated and cultivated on MEA, CYA, Creatine- usually attack grapes before harvest. Aside from losses in Sucrose agar (CREA) (Samson et al., 2010) and Yeast fruit, this species is regarded as the major producer of Extract agar (YES) (Samson et al., 2010). The Penicillium patulin, although this species produces many other toxic colonies were identified to species level according to Pitt metabolites such as citrinin, roquefortine C or and Hocking (2009) and Samson and Frisvad (2004). chaetoglobosins among others (Andersen, Smedsgaard and Frisvad, 2004). Mycotoxin production Toxinogenity of selected isolates was screened in in vitro Scientific hypothesis conditions by means of thin-layer chromatography (TLC) Some of the fungal species occurring on grapes and grape according to Samson et al. (2002), modified by Labuda products can produce mycotoxins, so species identification and Tančinová (2006). Extracellular metabolites – citrinin is critical to predicting the potential mycotoxin griseofulvin and patulin were carried out on YES agar and contamination of grapes and wine. intracellular cyclopiazonic acid, penitrem A, and roquefortin C on CYA agar. At 14 days of incubation, five MATERIAL AND METHODOLOGY agar plugs (4 mm diameter) were cut from the edge of Study area a colony (extracellular metabolites) or cut from a colony Village Vrbové is located in the Vrbovský subregion in (intracellular metabolites) from each Petri plate and placed the Small Carpathian wine region. The Small Carpathian in an Eppendorf tube. The plags were extracted in 500 µL wine region is the most extensive of the six wine regions of chloroform-methanol (2:1, v/v) (Reachem, Slovak in Slovakia (vineyards are covering 4175 hectares) and is Republic). The content of the tubes was stirred for 5 min located in the southwestern part of Slovakia (ÚKSÚP, by Vortex Genie ® 2 (MO BIO Laboratories, Inc. – 2019b). Vines have been grown on the south-facing slopes Carlsbad, CA, USA). The extract of liquid phase 30 µL of the Small Carpathian mountains in locality Záhorie for along with 10 µL of standards (Sigma, Germany) was more than three thousand years. This region has a medium transferred to the TLC plate (Alugram ® SIL G, Macherey climate and abundant moisture. – Nagel, Germany). The plate was put into TEF solvent Last year, as a whole, was extremely warm. The year (toluene:ethyl acetate:formic acid – 5:4:1, toluene – 2019 had the same average annual temperature in Mikrochem, Slovak Republic; ethyl acetate and formic Hurbanovo 12.42 °C as in 2018. This value is a record acid – Slavus, Slovak Republic). After elution, the plate high for Hurbanovo since the record began. During the was air-dried. The identification of the metabolites was whole year, 2019 was only one month of the territory done by comparison with metabolite standards. temperature below normal. It was May (Beránek and Cyclopiazonic acid was visible directly in daylight after Faško, 2020). spraying with the Ehrlich reagent as a violet-tailed spot. Penitrem A was visible after spraying with 20% AlCl in 3 60% ethanol and heating at 130 °C for 8 min as a dark blue Grape sampling spot. Roquefortin C was visible after spraying with A total of 13 samples were taken: 3 from red varieties Ce(SO4)2 x 4 H2O as an orange spot. Patulin detection was (Alibernet, Cabernet Sauvignon, and Blaufränkisch) and achieved by spraying with 0.5% methylbenzothiazolone 10 from white varieties (Palava, Green Veltliner, Seteasca hydrochloride (MBTH) (Merck, Germany) in methanol Regala, Chardonnay, Rheinriesling, Welschriesling, and heating at 130 °C for 8 min and then detected as Sauvignon, Pinot Blanc, Irsai Oliver, and Müller Thurgau). a yellow-orange spot under visible light. Citrinin was The sampling was conducted at the 2019 vintage, at the detected directly as an intense yellow‐green streak under end of September. Two diagonals crossing the vineyards ultraviolet light (365 nm) as well as griseofulvin, which were delimited, and five healthy and undamaged bunches was visible as a blue spot. from each diagonal were obtained. Each bunch was collected in a sterilized plastic bag and sent to the laboratory chilled on ice. Statistical analysis The obtained results were evaluated and expressed according to relative density (RD) and isolation frequency Mycological analysis (Fr). The relative density (%) is defined as the percentage Fifty berries were selected randomly
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